Salicylate improves macrophage cholesterol homeostasis via activation of Ampk

Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk β1-deficient (β1−/−) mice. Macrophages from Ampk β1−/− mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk β1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk β1−/−macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk β1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk β1−/− macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity

<i>In vivo</i> choline kinase inhibition during primary infection impairs systemic and intestinal RELMα and intestinal macrophage alternative activation.

A) Schematic of primary infection with 200 H. polygyrus (Hp) L3 larvae through oral gavage. Mice were treated intraperitoneally with vehicle (40% DMSO in PBS) or RSM-932a (3 mg/kg) every other day for 8 days starting on day 8 and sacrificed on day 17. B-C) Percentage of weights at start of vehicle or RSM injections at 8 DPI with H. polygyrus (B) or final weights at day 17 (C). Mixed-effects analysis with Tukey’s test for multiple comparisons (* p H. polygyrus-infected mice. n = 3–5 per group. Two-way ANOVA with Šídák’s test for multiple comparisons (* p ## p + (I) or RELMα+ (J) per DAPI+ cell. Unpaired t test (* p + (K) and RELMα+F4/80+ (L) per DAPI+ cell. n = 6. Two-way ANOVA with Šídák’s test for multiple comparisons (**** p < 0.0001). Schematics were created using BioRender.</p

Expression of activation markers in peritoneal macrophages during helminth infection.

A) Gating strategy of peritoneal cell populations. B) Enumeration of MHCIIhi and MCHIIlo large and small peritoneal macrophages among live PECs. n = 4–5, representative of 2 experiments. Unpaired t test (** p hi and MCHIIlo large and small peritoneal macrophages. Unpaired t test (*, p (TIF)</p

Graphical abstract.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.</div

Inhibiting choline metabolism drives pronounced changes in gene transcription in naïve and IL-4-polarized macrophages.

A) Schematic of bulk RNA-seq sample preparation. Heatmap of z-scores for select genes of interest. n = 3. B) Volcano plots of comparisons between Veh and HC3 or RSM in M[0] or M[IL-4]. Red dots represent genes with false discovery rate (FDR) 2 fold change (FC) 1. Select genes of interest are annotated. C-D) The top significantly up- or down-regulated genes shared in all comparisons in B) were analyzed by Enrichr-KG to identify common pathways. Schematics were created using BioRender.</p

IL-4 up-regulates choline metabolism in macrophages.

A) Schematic of lipidomics analysis. B-G) Total PC content (B), heatmap of PC species (C), SM content (D), PC(O) content (E), PC(P) content (F), or total LPC content (G) for M[0], M[IL-4], or M[LPS] expressed as nmol per 106 cells. n = 5 per polarization. Heatmap statistics are shown as fold change over the average of M[0]. One-way ANOVA with Dunnett’s test for multiple comparisons (* p 3H-choline into phospholipids over time. n = 5 per timepoint, representative of 3 experiments. Two-way ANOVA with Šídák’s test for multiple comparisons (** p 3H-choline over time in M[0] or M[IL-4]. n = 3 per timepoint, representative of 3 experiments. Linear regression F test (**** p Slc44a1 and Slc44a2 transcripts in M[0] or M[IL-4], normalized to Actb. n = 8 mice. K) Expression by Western blot of Slc44a1 and Slc44a2 in M[0] or M[IL-4]. Densitometry quantified relative to β-actin. n = 3, representative of 3 experiments. Unpaired t test (** p < 0.01). L) Expression of Slc44a1 by surface flow cytometry after different IL-4 stimulation times. Representative of 2 experiments. Schematics were created using BioRender.</p

Raw data file.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated upregulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.</div

Choline-containing phospholipid species in polarized macrophages.

A-I) Sum of phospholipid subclasses. A) Plasmenyl-lysophosphatidylcholines (LPC(P)), B) plasmanyl-LPCs (LPC(O)), C) platelet activating factors (PAF). D) D) Heatmap of sphingomyelin (SM), plasmanyl-PAFs (PC(O)-PAF), PC(O), PC(P), LPC, LPC(O), and LPC(P). Heatmap statistics are shown as fold change over the average of M[0]. n = 5. One-way ANOVA with Dunnett’s test vs M[0] for multiple comparisons (* p (TIF)</p